Conola variety IMC 02 with reduced linolenic acid

ABSTRACT

A canola line has been stabilized to produce seeds having an α-linolenic acid content of less than that of generic canola oil, more preferably lass than or equal to about 7% α-linolenic acid relative to total fatty acid content of said seed and a total glucosinolate content of less than 18 μmol/g of defatted meal, more preferably less than or equal to about 15 μmol/g of defatted meal. This canola line has reduced sulfur content of less than or equal to 3.0 ppm, improved sensory characteristics and increased oxidative stability.

TECHNICAL FIELD

This invention relates to improved canola seeds, plants and oil havingadvantageous properties, that is, a low glucosinolates content and avery low α-linolenic acid (C_(18:3)) content, which produce an oil withlow sulfur content, improved sensory characteristics and oxidativestability.

1. BACKGROUND

A need exists for an improved vegetable oil with a significantlyextended shelf life and greater heat stability relative to genericcanola oil and a positive nutritional contribution to animal, includinghuman, diets.

Canola oil has the lowest level of saturated fatty acids of allvegetable oils. “Canola” refers to rapeseed (Brassica) which has anerucic acid (C_(22:1)) content of at most 2 percent by weight based onthe total fatty acid content of a seed, preferably at most 0.5 percentby weight and most preferably essentially 0 percent by weight and whichproduces, after crushing, an air-dried meal containing less than 30micromoles (μmol) per gram of defatted (oil-free) meal. These types ofrapeseed are distinguished by their edibility in comparison to moretraditional varieties of the species.

As consumers become more aware of the health impact of lipid nutrition,consumption of canola oil in the U.S. has increased. However, genericcanola oil cannot be used in deep frying operations, an importantsegment of the food processing industry.

Canola oil extracted from natural and previously commercially usefulvarieties of rapeseed contains a relatively high (8%-10%) α-linolenicacid content (C_(18:3)) (ALA). This trienoic fatty acid is unstable andeasily oxidized during cooking, which in turn creates off-flavors of theoil (Gailliard, 1980, Vol. 4, pp. 85-116 In: Stumpf, P. K., ed., TheBiochemistry of Plants, Academic Press, New York). It also develops offodors and rancid flavors during storage (Hawrysh, 1990, Stability ofcanola oil, Chapter 7, pp. 99-122 In: F. Shahidi, ed. Canola andRapeseed: Production, Chemistry, Nutrition, and Processing Technology,Van Nostrand Reinhold, N.Y.). One such unsatisfactory species heretoforehas been Brassica napus, i.e., spring canola, a type of rapeseed.

It is known that reducing the α-linolenic content level by hydrogenationincreases the oxidative stability of the oil. Hydrogenation is routinelyused to reduce the polyunsaturates content of vegetable oils, therebyincreasing its oxidative stability. The food industry has usedhydrogenation to raise the melting-point of vegetable oils, producingoil-based products with textures similar to butter, lard and tallow.Trans isomers of unsaturated fatty acids are commonly produced duringhydrogenation. However, the nutritional properties of trans fatty acidsmimic saturated fatty acids, thereby reducing the overall desirabilityof hydrogenated oils (Mensink. et al., New England J. MedicineN323:439-445, 1990; Scarth, et al., Can. J. Pl. Sci., 68:509-511, 1988).Canola oil produced from seeds having a reduced α-linolenic acid contentwould be expected to have improved functionality for cooking purposeswith improved nutritional value, and therefore have improved value as anindustrial frying oil.

However, in general, very little variation exists for α-linolenic acidcontent in previously known canola quality B. napus germplasm (Mahler etal., 1988, Fatty acid composition of Idao Misc. Ser. No. 125). Lineswith levels of α-linolenic acid lower than that of generic canola oilare known, but have sensory, genetic stability, agronomic or othernutritional deficiencies. For example, Rakow et al. (J. Am. Oil Chem.Soc., 50:400-403, 1973), and Rakow (Z. Pflanzenzuchtg, 69:62-82, 1973),disclose two α-linolenic acid mutants, M57 and M364, produced bytreating rapeseed with X-ray or ethylmethane sulfonate. M57 had reducedα-linolenic acid while M364 had increased α-linolenic acid. However, theinstability of the fatty acid traits between generations wasunacceptable for commercial purposes.

Brunklaus-Jung et al. (Pl. Breed., 98:9-16, 1987), backcrossed M57 andother rapeseed mutants obtained by mutagenic treatment to commercialvarieties. BC₀ and BC₁ of M57 contained 29.4-33.3% of linoleic acid(C_(18:2)) and 4.9-10.8% of α-linolenic acid (C_(18:3)). The oleic acid(C_(18:1)) content was not reported, but by extrapolation could not haveexceeded 60%.

Four other lower α-linolenic acid canola lines have been described.Stellar, reported by Scarth et al. (Can. J. Plant Sci., 68:509-511,1988), is a Canadian cultivar with lower α-linolenic acid (also 3%)derived from M57. Its α-linolenic acid trait was generated by seedmutagenesis. S85-1426, a Stellar derivative with improved agronomiccharacteristics, also has lower (1.4%) α-linolenic acid (Report of 1990Canola/Rapeseed Strain Test A, Western Canada Canola RapeseedRecommending Committee). IXLIN, another lower α-linolenic acid (1.8%)line described by Roy et al. (Plant Breed., 98:89-96, 1987), originatedfrom an interspecific selection. EP-A 323 753 (Allelix) discloses rapeplants, seeds, and oil with reduced α-linolenic acid content linked tolimitations in the content of oleic acid, erucic acid, andglucosinolate.

Another nutritional aspect of rapeseed, from which canola was derived,is its high (30-55 μmol/g) level of glucosinolates, a sulfur-basedcompound. When the foliage or seed is crushed, isothiocyanate esters areproduced by the action of myrosinase on glucosinolates. These productsinhibit synthesis of thyroxine by the thyroid and have otheranti-metabolic effects (Paul et al., Theor. Appl. Genet. 7:706-709,1986). Brassica varieties with reduced glucosinolates content (<30μmol/g defatted meal) were developed to increase the nutritional valueof canola meal (Stefansson et al., Can. J. Plant Sci. 55:343-344, 1975).Meal from an ultra-low glucosinolates line, BC86-18, has 2 μmol/g totalglucosinolates and significantly improved nutritional quality comparedto generic canola meal (Classen, Oral presentation, GCIRC EighthInternational Rapeseed Congress, Saskatoon, Saskatchewan, Jul. 9-11,1991). Neither its fatty acid composition nor its seed glucosinolatesprofile is known.

There remains a need for an improved canola seed and oil with very lowα-linolenic levels in the oil and low glucosinolates in the seed tosignificantly reduce the need for hydrogenation. The α-linolenic contentof such a desirable oil would impart increased oxidative stability,thereby reducing the requirement for hydrogenation and the production oftrans fatty acids. The reduction of seed glucosinolates wouldsignificantly reduce residual sulfur content in the oil. Sulfur poisonsthe nickel catalyst commonly used for hydrogenation (Koseoglu et al.,Chapter 8, pp. 123-148, In: F. Shahidi, ed. Canola and Rapeseed:Production, Chemistry, Nutrition, and Processing Technology, VanNostrand Reinhold, N.Y., 1990). Additionally, oil from a canola varietywith low seed glucosinolates would be less expensive to hydrogenate.

2. SUMMARY OF THE INVENTION

This invention comprises a Brassica napus canola yielding seed having atotal glucosinolates content of about 18 μmol/g or less of defatted,air-dried meal; the seed yielding extractable oil having 1) anα-linolenic acid content of about 7% or less relative to total fattyacid content of the seed, and 2) a very low sulfur content of less thanor equal to 3.00 ppm. The invention also includes a Brassica napusyielding canola oil having, when hydrogenated, a significantly reducedoverall room-odor intensity relative to the overall room-odor intensityof generic canola oil. The new variety more particularly yieldsnon-hydrogenated oil significantly reduced in fishy odor relative to thefishy odor of generic canola oil, such odor being characteristic ofBrassica seed oil. The seed of such canola variety has an α-linolenicacid content of less than or-equal to 7%, more preferably less than orequal to about 4.1% α-linolenic acid (C_(18:3)) relative to total fattyacid content of said seed and a total glucosinolates content of lessthan 18 μmol/g, more preferably less than or equal to about 15 μmol/gand most preferably less than or equal to 13 μmol/g and belongs to aline in which these traits have been stable for both the generation towhich the seed belongs and that of its parent.

This invention further includes processes of making crosses using IMC 01as at least one parent of the progeny of the above-described seeds andoil derived from said seeds.

This invention further comprises a seed designated IMC 01 deposited withthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md., USA 20852 and bearing accession number ATCC 40579, the progeny ofsuch seed and oil of such a seed possessing the quality traits ofinterest.

DETAILED DESCRIPTION OF THE INVENTION

A spring canola (Brassica napus L.) variety was developed with improvedsensory characteristics and oxidative stability in the seed oil. Thisvariety, designated IMC 01, has very low levels of α-linolenic acid(CH₃CH₂CH═CHCH₂CH═CHCH₂CH═CH (CH₂)₇COOH) in the seed oil and very lowlevels of glucosinolates in the seed. The oil produced from the seed ofthis variety has very low levels of sulfur and was shown to havesignificantly improved sensory characteristics over generic canola oils.The IMC 01 is a line in which these traits have been stabilized for boththe generation to which the seed belongs and that of its parentgeneration. Particularly desirable lines of this invention from anagronomic point of view can be derived by conventionally crossing linesproducing seeds meeting the definitions of this invention withagronomically well-proven lines such as Westar.

In the context of this disclosure, a number of terms are used. As usedherein, a “line” is a group of plants that display little or no geneticvariation between individuals for at least one trait. Such lines may becreated by several generations of self-pollination and selection, orvegetative propagation from a single parent using tissue or cell culturetechniques. As used herein, the terms “cultivar” and “variety” aresynonymous and refer to a line which is used for commercial production.“Stability” or “stable” means that with respect to the given component,the component is maintained from generation to generation and,preferably, at least three generations at substantially the same level,e.g., preferably ±15%, more preferably ±10%, most preferably ±5%. Thestability may be affected by temperature, location, stress and the timeof planting. Comparison of subsequent generations under field conditionsshould produce the component in a similar manner. “Commercial Utility”is defined as having good plant vigor and high fertility, such that thecrop can be produced by farmers using conventional farming equipment,and the oil with the described components can be extracted from the seedusing conventional crushing and extraction equipment. To be commerciallyuseful, the yield, as measured by both seed weight, oil content, andtotal oil produced per acre, is within 15% of the average yield of anotherwise comparable commercial canola variety without the premium valuetraists grown in the same region. “Agronomically elite” means that aline has desirable agronomic characteristics such as yield, maturity,disease resistance, standability. The amount of fatty acids, such asoleic and linolenic acids, that are characteristic of the oil isexpressed as a percentage of the total fatty acid content of the oil.“Saturated fatty acid” refers to the combined content of palmitic acidand stearic acid. “Polyunsaturated fatty acid” refers to the combinedcontent of linoleic and α-linolenic acids. The term “shortening” refersto an oil that is a solid at room temperature. The term “room odor”refers to the characteristic odor of heated oil as determined using theroom-odor evaluation method described in Mounts (J. Am. Oil Chem. Soc.,56:659-663, 1979). “Generic canola oil” refers to a composite oilextracted from commercial varieties of rapeseed currently known as ofthe priority date of this application, which varieties generallyexhibited at a minimum 8-10% α-linolenic acid content, a maximum of 2%erucic acid and a maximum of 30 μmol/g total glucosinolate level. Theseed from each growing region is graded and blended at the grainelevators to produce an acceptably uniform product. The blended seed isthen crushed and refined, the resulting oil being sold for use. Table Ashows the distribution of canola varieties seeded as percentage of allcanola seeded in Western Canada in 1990. TABLE A Distribution of CanolaVarieties Grown in Western Canada in 1990 Percent of Canola VarietySeeded Area B. campestris Candle 0.4 Colt 4.4 Horizon 8.5 Parkland 2.5Tobin 27.1 B. napus Alto 1.1 Delta 0.9 Global 0.9 Legend 18.2 Pivot 0.1Regent 0.5 Stellar 0.2 Tribute 0.4 Triton 0.7 Triumph 0.2 Westar 29.5Others 4.4Source: Quality of Western Canadian Canola—1990 Crop Year. Bull. 187,DeClereg et al., Grain Research Laboratory, Canadian Grain Commission,1404-303 Main Street, Winnipeg, Manitoba, R3C 3G8.

IMC 01 is a very low α-linolenic acid (<4.1% C_(18:3)) line selectedduring an extensive germplasm screening effort. Its parentage isunknown. IMC 01 was self-pollinated and selected for low α-linolenicacid (<4.1%) over four consecutive generations. At each generation,seeds from individually pollinated plants were analyzed for fatty acidcomposition. Data showed no genetic segregation for α-linolenic acidcontent over five generations of self-pollinations (Table I). Breederseed was derived from a bulk seed increase of selected plants from thefourth self-crossed generation. TABLE I Fatty Acid Composition of IMC 01Over Five Generations DATE OF PERCENT COMPOSITION ANALYSIS C_(16:0)C_(18:0) C_(18:1) C_(18:2) C_(18:3) November 1987 4.1 1.9 64.1 25.7 1.9August 1988 4.6 2.3 72.6 14.4 2.0 January 1989 4.9 1.5 60.4 25.8 2.5April 1989 4.8 1.8 64.3 21.4 4.0 October 1989 4.3 2.1 64.1 24.8 2.0

IMC 01 was planted in replicated field trials in North Dakota, SouthDakota, Minnesota, Washington, Idaho and Montana in 1989 and 1990, underboth irrigated and nonirrigated conditions. These tests showed that theα-linolenic acid content of IMC 01 was sensitive to temperature (TableII). This was further supported by growing IMC 01 under controlledtemperature conditions in growth chambers. Whether or not the observedtemperature sensitivity of IMC 01 is common to other low α-linolenicacid canola lines is unknown.

A temperature effect on fatty acid compositions has been widely reportedin plants, especially oilseed crops (Rennie et al., J. Am. Oil Chem.Soc., 66:1622-1624, 1989). These reports describe general temperatureeffects on fatty acid composition.

Changes in fatty acid content in seed oil under cool temperatures havebeen documented in plants such as soybean, peanut and sunflower(Neidleman, In: Proceedings of the World Conference on Biotechnology forthe Fats and Oils Industry, Applewhite, T. H., ed., pp. 180-183. Am.Oil. Chem. Soc. 1987). TABLE II α-Linolenic acid Content of IMC 01 inProduction in 1990 α-LINOLENIC PRODUCTION {overscore (X)} TEMPERATUREDURING ACID REGION SEED MATURATION CONTENT Eastern Washington 74° F.1.9% Eastern Washington 74° F. 2.0% Northern Idaho 70° F. 3.0% NorthernIdaho 67° F. 2.9% Eastern Idaho 62° F. 3.5% Southern Montana 66° F. 4.1%Central Montana 67° F. 4.0%

In addition to very low α-linolenic acid, IMC 01 is also characterizedby very low levels of glucosinolates. Glucosinolates are sulfur-basedcompounds common to all Brassica seeds. Glucosinolates exist inaliphatic or indolyl forms. Aliphatic glucosinolates can be analyzed viagas chromatography (GC) (Daun, Glucosinolate analysis of rapeseed(canola), Method of the Canadian Grain Commission, Grain ResearchLaboratory, Canadian Grain Commission, Winnipeg, 1981). Indolylglucosinolates have only recently been analyzed via high performanceliquid chromatograph (HPLC). Prior to the adoption of the HPLC method,total glucosinolates were calculated by multiplying the aliphaticglucosinolates by a correction factor. Canola quality in the seed isdefined as having <30 μmol/g of glucosinolates in the defatted meal.

IMC 01 and Westar were tested in five locations in southeastern Idaho in1990. Three of the locations were irrigated (I) and two were dryland (D)conditions. Table IIIa shows the difference in total aliphaticglucosinolate between IMC 01 and Westar grown at these locations. Thealiphatic glucosinolate values are reported as μmol/gm of defatted meal.

The aliphatic glucosinolate content of IMC 01 by location wasconsistently lower and more stable than that of Westar at all locationstested. The average glucosinolate contents of IMC 01 was 4.9 μMol/gmwhile Westar was 13.3 μmol/gm. A Least Significant Difference (LSD) testwas used to determine if the two were significantly different at alllcoations. IMC 01 was found to be significantly different from Westar ata level of P<0.05.

HPLC analysis of IMC 01 vs Westar, the most widely grown spring canolavariety in North America, shows that IMC 01 has much lower levels ofaliphatic glucosinolates (Table III). No significant differences existfor indolyl glucosinolates. Glucosinolates content is also subject toenvironment influence, e.g., sulfur fertility and drought stress.However, IMC 01 consistently had the lowest and the most stablealiphatic glucosinolates levels at all locations tested (Table IV). Thelocations tested differ in altitude, temperature, fertility, irrigation,and other cultural practices. Among the low α-linolenic canola lines forwhich glucosinates analysis have been performed, IMC 01 has the lowestlevel of total seed glucosinolates (Table V). TABLE III GlucosinolatesProfiles of IMC 01 and Westar Varieties GLUCOSINOLATES (μmol/g) IMC 01WESTAR 3-butenyl 1.2 4.2 4-pentenyl 0.1 0.2 2-OH-3-butenyl 3.1 7.02-OH-4-pentenyl 0.9 0.4 Total Aliphatics 5.3 11.8 4-OH-3-indolylmethyl6.2 6.1 3-indolylmethyl 0.8 1.0 4-methoxyindolyl 0.1 tr 1-methoxyindolyl0.1 0.2 Total Indolyls 7.2 7.3 Total Glucosinolates 12.5 19.1

TABLE IV Aliphatic Glucosinolates of IMC 01 and Westar over DifferentEnvironments in Southeastern Idaho ALIPHATIC GLUCOSINOLATES CONTENT(μmol/g) LOCATION* IMC 01 WESTAR Newdale - I 4.7 13.0 Soda Springs - D6.3 9.9 Tetonia - D 5.0 13.5 Tetonia - I 3.7 13.3 Shelley - I 5.0 17.0Average 4.9 13.3 Standard Deviation 0.93 2.51*I = Irrigated,D = Dryland

TABLE V Glucosinolates Content of Low α-Linolenic acid Canola Varietiesand Westar GLUCOSINOLATES (μmol/g) ALIPHATIC INDOLYL TOTAL IMC 01 5.37.2 12.5 Stellar 5.2 19.5 24.7 S85-1426 7.9 13.4 21.3 Westar 11.8 7.319.1

IMC 01 was produced, using normal production practices for springcanola, in Idaho and North Dakota in 1988, in Idaho, Washington Stateand Montana in 1989, in Idaho, Washington State, Montana, Oregon, andWyoming in 1990. When grown in suitable environments, where the averagedaily temperature (high temperature plus low temperature divided by 2)exceeds 20° C., the oil contains <4.1% α-linolenic acid. As an example,a normal fatty acid profile was produced at Casselton, North Dakota. Thecrop produced in Ashton, Id., was subject to extremely cool conditionsand had higher levels of α-linolenic acid. The crops obtained from thefield tests were crushed and processed to produce refined, bleached anddeodorized (RBD) canola oil at the Protein, Oil, Starch (POS) PilotPlant in Saskatoon, Saskatchewan. A method of bleaching canola oil isprovided in the AOCS' Recommended Practice Cc 8a-52 (AOCS Methods andStandard Practices, 4th Edition (1989)). A method for the refining ofcrude oils is provided in the AOCS Practice Cc 9a-52 (AOCS Methods andStandard Practices, 4th Edition (1989)). The oils were tested at theVegetable Oil Research Laboratory, U.S.D.A./Northern Regional ResearchCenter, for organoleptic and sensory characteristics.

Testing to assure that desirable sensory characteristics are obtained inthe Brassica napus variety was essential. The evaluation of odors hasbeen conducted in a variety of ways on low α-linolenic acid canola oils.The testing methods are based on the fact that vegetable oils emitcharacteristic odors upon heating. For example, Prevot et al. (J. Amer.Oil Chemists Soc. 67:161-164, 1990) evaluated the odors of a Frenchrapeseed, “Westar”, and “low linolenic” canola oils in a test whichattempted to reproduce domestic frying conditions. In these evaluationsthe test oils were used to fry potatoes and the odors were evaluated bya test panel. The odor tests showed that the “low linolenic”(approximately 3%) line had a significantly higher (more acceptable)odor score than the French rapeseed and “Westar” lines, which were verysimilar to each other. Eskin et al. (J. Amer. Oil Chemists Soc.66:1081-1084, 1989) evaluated the odor from canola oil with a lowlinolenic acid content, a laboratory deodorized sample, and acommercially deodorized sample by sniffing in the presence of the oilitself. These studies demonstrated that a reduction in the linolenicacid content of canola oil from 8-9% to 1.6% reduced the development ofheated odor at frying temperatures. However, the odor of the lowlinolenic acid oil was still unacceptable when heated in air to amajority of the panelists, suggesting that low linolenic acid alone isnot sufficient to guarantee acceptable odor.

Mounts (J. Am. Oil Chem. Soc., 56:659-663, 1979) describe a distinctroom-odor evaluation method that is used to reproducibly assess the odorcharacteristics of a cooking oil upon heating. This is the evaluationmethod of choice owing to its reproducibility and its approximation ofodors emitted upon heating the oil. In this method, the oil is heated ina separate chamber and the odor pumped into the room containing thetrained evaluators. As noted elsewhere, where the term “room-odor” isused herein, it refers to this method of Mounts. This method is distinctfrom earlier described tests where the oil and evaluator are within thesame room. Such same room testing is referred to as “uncontrolled benchtop odor tests” and is considered less accurate and less reliable thanthe Mounts' room odor evaluation method.

The room-odor characteristics of cooking oils can be reproduciblycharacterized by trained test panels in room-odor tests (Mounts, J. Am.Oil Chem. Soc. 56:659-663, 1979). A standardized technique for thesensory evaluation of edible vegetable oils is presented in AOCS'Recommended Practice Cg 2-83 for the Flavor Evaluation of Vegetable Oils(Methods and Standard Practices of the AOCS, 4th Edition (1989)). Thetechnique encompasses standard sample preparation and presentation, aswell as reference standards and method for scoring oils. When heated,generic canola oil has reduced stability and produces offensive roomodors. Refined-Bleached-Deodorized (RBD) canola oil is characterized bya fishy flavor in such tests. This characteristic is commonly ascribedto its high polyunsaturated fatty acid content, particularly α-linolenicacid, relative to other vegetable oils. The individual fragrance notes(odor attributes) of the oils are evaluated by Least SignificantDifference Analysis. Notes which differ by greater than 1.0 can bereproducibly measured by a sensory panel. In these tests, IMC 01 oilexpressed significantly reduced levels of the offensive odors (TableVI). TABLE VI Room Odor Intensity of IMC 01 and Generic Canola Oil ODORATTRIBUTES IMC 01 GENERIC CANOLA OIL Overall  4.6^(a) 7.4^(b) FriedFoods 1.8 3.5 Doughy 1.0 0 Fishy  0^(a)   5.5^(b) Burnt  0^(a)   0.9^(b)Acrid  0^(a)   2.3^(b) Woody/Cardboard 1.9 0 Hydrogenated 0   0Pastry/Sugary 0   0 Waxy 0   0 Chemical 0   00 = None;10 = Strong.Scores with different superscript letters are significantly different (P< 0.05). Least Significant# Difference for individual odor notes is 1.0. Differences greater than1.0 can be reproducibly measured by room-odor analysis.

Due to its relatively low stability, canola oil is often hydrogenatedfor frying. However, hydrogenation produces a characteristic(hydrogenated) room odor which is unacceptable to food manufacturers.Surprisingly, hydrogenated IMC 01 oil also has reduced levels of thecharacteristic hydrogenated room odor (Table VII). Table VII shows thatthe overall room-odor intensity of hydrogenated IMC 01 is significantlyless than that of hydrogenated generic oil as indicated by a differenceis scores of greater than 1.0 in standardized flavor evaluation trials.TABLE VII Room Odor Intensity and Individual Odor Descriptions forHydrogenated Canola Oils HYDROGENATED HYDROGENATED IMC 01 HYDROGENATED,IMC 01 SHORTENING GENERIC SHORTENING (2% (6.8% CANOLA α-LINOLENICα-LINOLENIC ODOR ATTRIBUTE SHORTENING ACID) ACID) overall Intensity6.6^(b) 3.8^(a) 3.9^(a) Fried Food 2.7 1.1 1.5 Doughy 1.2 0.6 0.8 Fishy0.6 0 0 Burnt 0.5 0 0 Acrid 0.8 0 0 Hydrogenated 3.2 1.8 2.3 Waxy 0.50.6 0. Other 4.5 2.4 2.8 rubbery fruity fruity flowery smoky floweryweedy sweet soapy pastry0 = None;10 = Strong.Scores with different superscript letters are significantly different (P< 0.05). Least Significant# Difference for individual odor notes is 1.0. Differences greater than1.0 can be reproducibly measured by room-odor analysis.

IMC 01 produces an oil which has improved sensory characteristics. Suchimprovements have been predicted for low α-linolenic acid canola oils(Ulrich et al., J. Am. Oil Chem. Soc., 8:1313-1317, 1988). However, theimproved sensory characteristics of IMC 01 appears not to be relatedsolely to its low α-linolenic acid content. Surprisingly, IMC 01 canolaoils with both high and low levels of α-linolenic acid showed similardegrees of improvement. Sensory tests have shown that IMC 01 oilmaintains its improved quality at both 2% and 6.8% α-linolenic acid.

The very low glucosinolates characteristic of IMC 01 seed is believed tocontribute to the improved sensory characteristic of IMC 01 oil.Glucosinolates in the seed are converted to sulfur compounds. Most ofthe sulfur breakdown products remain in the meal, but some inevitablycontaminate the oil. Lower levels of glucosinolates in the seed arebelieved to result in lower sulfur content in the oil, and this isbelieved to reduce the objectionable odor characteristics of canola oil(Abraham et al., J. Am. Oil Chem. Soc., 65:392-395, 1988). An analysisof the sulfur content of IMC 01 oil and several generic canola oils hasbeen performed. IMC 01 oil has approximately one-third the sulfurcontent of leading generic canola oils (Table VIII). TABLE VIII SulfurContent of Canola Oils Canola Oils Sulfur Content Acme Brand Canola Oil3.8 ppm Hollywood Brand Canola Oil 3.8 ppm Puritan Brand Canola Oil 3.9ppm IMC 01 Canola Oil 1.3 ppm

The biochemical, molecular and genetic mechanisms responsible for theroom-odor quality of vegetable oils are not fully understood.Improvements in vegetable oil processing technology, i.e., preferentialremoval of sulfur during processing, less abusive oil extractionprocedures, minimal processing, gentler deodorization, etc., may improvethe overall quality of vegetable oils, including both sensory andfunctional characteristics (Daun et al., J. Am. Oil Chem. Soc.,53:169-171, 1976). IMC 01 will benefit from any such processingimprovements, and will maintain its improved sensory characteristicsover generic canola oil under equivalent processing conditions.

IMC 01 is true breeding as are its progeny. The traits responsible forreduced α-linolenic acid and reduced total glucosinolates in the seedwhich yield an oil low in sulfur having improved sensory characteristicshave a genetic basis. The data presented herein show that these traitsare stable under different field conditions. These traits can be removedfrom the IMC 01 background and are transferred into other backgrounds bytraditional crossing and selection techniques.

Crosses have been made with IMC 01 as one parent to demonstrate that thesuperior IMC 01 quality/sensory traits are transferred along with thesuperior agronomic traists of another patent such as the Canadian canolaline, Westar, into descendents. The parent to which IMC 01 is crossed ischosen on the basis of desirable characteristics such as yield,maturity, disease resistance, and standability. Conventional breedingtechniques employed in such crossings are well known by those skilled inthe art. Thus, a method of using the IMC 01 Brassica napus is to crossit with agronomically elite lines to produce plants yielding seedshaving the characteristics listed above.

The general protocol is:

-   -   a. cross IMC 01 to a selected parent;    -   b. produce a “gametic array” using microsphores of the F₁ plants        to produce dihaploid (DH) individuals;    -   c. field trial DH₂ individuals for yield and select from IMC 01        α-linolenic acid and glucosinolate levels; and    -   d. test selected individuals for oil quality using RBD oil.

Example 3 is a specific example of such work to develop descendents toIMC 01 which retain the desirable quality traits. The data of Example 3show that the quality traits of IMC 01 are heritable in such crosses.

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that this Example,while indicating preferred embodiments of the invention, is given by wayof illustration only. From the above discussion and this Example, oneskilled in the art can ascertain the essential characteristics of thisinvention, and without departing from the spirit and scope thereof, canmake various changes and modifications of the invention to adapt it tovarious usages and conditions.

EXAMPLE1

IMC 01, originally designated DNAP #336, was grown in a greenhouse inCinnaminson, N.J., over several seasons to select for a stable, very lowα-linolenic line. Day/night temperatures from August through December inthe greenhouse averaged 80° F./65° F. with fluctuations of ±5° F., 75°F./65° F. from January through April, and 85° F./65° F. from Marchthrough July. The plants were grown in 1-gallon pots under natural daylength, except from October through May when the plants received 14hours of supplemental lighting. Flowering racemes were covered withpaper bags to prevent cross-pollination, and gently shaken to induceseed set. Watering was decreased as pods reached maturity.

For field testing, IMC 01 was planted in multi-location trials andproduction plots in Montana, Idaho and Washington. The trials wereplanted in a completely randomized block design with four replications.Each block contained eight plots of 6 meters by 8 rows. IMC 01 was alsoplanted in large acreages (>25 acres) according to standard agronomicprocedures for spring canola production, with a minimum ½-mile isolationfrom other Brassica napus crops. Depending on location, the fieldswere-planted in April or May, and harvested in August or September.Plantings were made on dryland, following both fallow or recrop, orunder irrigation. Mature pod samples were taken following swathing forchemical analysis.

For fatty acid analysis, 10-50 seed samples were ground in 15-mLpolypropylene tubes and extracted in 1.2 mL 0.25 N KOH in 1:1ether/methanol. The sample was vortexed for 10 sec and heated for 60 secand in 60° C. water bath. Four mL of saturated NaCl and 2.4 mL ofiso-octane were added, and the mixture was vortexed again. After phaseseparation, 600 μL of the upper organic phase was pipetted intoindividual vials and stored under nitrogen. One μL sample was injectedinto a Supelco SP-2330 fused silica capillary column (0.25 mm ID, 30 mlength, 0.20 μm df, Bellfonte, Pa.).

The gas chromatograph was set at 180° C. for 5.5 min, then programmedfor a 2° C./min increase to 212° C., and held at this temperature for1.5 min. Chromatography settings were: Column head pressure−15 psi,Column flow (He)−0.7 mL/min, Auxiliary and Column flow−33 mL/min,Hydrogen flow=33 mL/min, Air flow−400 mL/min, Injector temperature−250°C., Detector temperature−300° C., Split vent−1/15.

A standard industry procedure for HPLC analysis of glucosinolates wasused to analyze the glucosinolates composition of the seed (Daun et al.,In: Glucosinolate Analysis of Rapeseed (Canola). Method of the CanadianGrain Commission, Grain Research Laboratory, 1981).

IMC 01 seed was harvested and processed to produce refined, bleached anddeodorized (RBD) oil. Some oil was hydrogenated after refining,bleaching and deodorization, then redeodorized.

Before extraction, the seed was tempered to adjust the moisture contentto 9% and flaked to 0.38 to 0.64 cm in a ribbon blender. The flakes werecooked in a stack cooker at 82.8° C. for 30 min (8.5% moisture) andpre-pressed with vertical and horizontal bar spacings set to 0.031 cm,vertical shaft speed at 40 rpm and horizontal shaft at 25 rpm. The presscake was extracted in a Crown Model 2 extractor at 37.3 kg and hexaneextracted with a 2:1 solvent to solids ratio.

The crude oil was alkali refined at 65° C.-70° C. for 30 min with 0.2%to 85% phosphoric acid, then mixed with sodium hydroxide to neutralizefree fatty acids. Soaps were removed with a water wash (65° C. water, 5min) and the oils bleached with 0.75% each by weight of Clarion andActicil bleaching earths for 30 min to remove color bodies. Theresulting oil contained no peroxides, 0.08% free fatty acids, and had aGardner color of 10-.

The oil was continuously deodorized at 265° C. at 300 kg/h. The steamrate was 1% of feed rate. The deodorized oil was preheated to 68-72° C.prior to deaeration. RBD oil was stored in food grade plastic drums orpails at 4° C. under nitrogen prior to testing.

For hydrogenation, RBD oil was heated to 350° F. under vacuum in astainless steel pressure reactor. A 0.5% sulfur-poisoned nickelcatalyst, Englehardt SP-7, was added to the oil at 80.1° C., andhydrogen gas was introduced at 40 psi. Periodic samples were analyzeduntil an oil with a 30.5° C. melting point was achieved. Thehydrogenated oil was redeodorized and stored by the methods describedpreviously.

The RBD and hydrogenated oil samples were analyzed for room-odorcharacteristics by a trained test panel in comparison with a generic,commercially available RBD canola oil (Proctor & Gamble) and generic,commercially available hydrogenated canola shortening as describedpreviously. The testing protocol used is described in Mounts (J. Am. OilChem. Soc. 56:659-663, 1979) which is hereby incorporated by reference.The testing controlled for temperature of the oil, distance from the oiland room volume, and required that the oil was heated in a separatechamber and pumped into the room containing the trained panelist.

Specifically, room odor profiles of IMC 01 and a generic canola oil wereobtained as follows:

A. Room Odor Protocol

A 150 mL sample of the selected oil was heated to 190° C. for 30 minbefore the start of each panel test. The oil was maintained at thistemperature throughout each session. For each session a fresh oil samplewas used.

Panelists visited each odor room for approximately 15 sec. A five minrest was required between visits. Visitation to each odor room wasrandomized among the panelists.

The trained panelists judged the room odor for intensity of odor,quality of odor, and odor attributes. The intensity was ranked as: 0-4weak, 5-7 moderate, and 8-10 strong. The quality of odor was judged as:0-1 bad, 2-3 poor, 4-6 fair, 7-8 good, and 9-10 excellent. The odorattributes were ranked as: 0-1 bland, 2-4 weak, 5-7 moderate, and 8-10strong. The flavor attributes were fried, painty, fishy, hydrogenated,burnt, cardboard, metallic, rubbery, waxy, buttery, and nutty.

B. Generic Oil—IMC 01 Profile Comparison

A generic, commercially available canola oil (Proctor & Gamble) was usedin the IMC room odor tests as the standard or generic canola oil. In acomparative test, the standard canola oil was significantly (P<0.05)higher in room odor intensity than IMC 01 (Table IX). The standardcanola oil odor was of “moderate” intensity while the IMC 01 wasconsidered “weak”. The overall quality of the IMC 01 room odor wassignificantly (P<0.05) better than the standard canola oil. The standardcanola oil had significantly (P<0.05) higher intensities for fried,painty, and cardboard odors than the IMC 01 oil. TABLE IX Room OdorProfile of Generic (Proctor & Gamble) and IMC 01 Oil Evaluation* IMC 01Generic A. Intensity 3.4^(a) 5.2^(b) B. Quality 5.8^(a) 4.9^(b) C. OdorAttributes Fried 1.9^(a) 2.9^(b) Painty 0.4 1.3 Fishy 0.8^(a) 1.9^(b)Hydrogenated 1.1 0.6 Rancid 0.7 0.9 Burnt 0.8 1.4 Cardboard 0.1^(a)1.5^(b) Metallic 0.5 0.1 Rubbery 0.0 0.0 Waxy 0.6 0.0 Buttery 0.7 0.3*The “intensity” was ranked as: 0-4 weak, 5-7 moderate, and 8-10 strong.The “quality” of odor was judged as: 0-1 bad, 2-3 poor, 4-6 fair, 7-8good, and 9-10 excellent. The “odor# attributes” were ranked as: 0-1 bland, 2-4 weak, 5-7 moderate, and8-10 strong.Scores with different superscript letters are significantly different (P< 0.05).

Pilot plant-processed samples of Example 2 generic canola (low erucicacid rapeseed) oil and oil from IMC 01 canola with the fatty acidcompositions modified by mutation breeding and/or hydrogenation wereevaluated for frying stability α-linolenic acid contents were 10.1% forgeneric canola oil, 1.7% for canola modified by breeding (IMC 01) and0.8% and 0.7% for IMC 01 oils modified by breeding and hydrogenation.The IMC 01 modified oils had significantly (P<0.05) less room odorintensity that the generic canola oil after initial heating tests at190° C. as judged by a sensory panel under conditions of AOCS Cg 2-83.The generic canola oil had significantly higher intensities for fishy,burnt, rubbery, smoky, and acrid odors than the modified oils. Foamheights of the modified oils were significantly (P<0.05) less than thoseof the generic oil after 20, 30 and 40 hrs of heating and frying at 190°C. The flavor quality of french fried potatoes was significantly(P<0.05) better for all the potatoes fried in modified oils than thosefried in generic canola oil. The potatoes fried in generic canola oilwere described by the sensory panel as fishy. No off-flavors weredetected in potatoes fried in the modified oils.

EXAMPLE 3

This Example demonstrates that the traits of very low α-linolenic acidand very low glucosinolate content are transferred to IMC 01 progeny.

The pre-production will be crushed and the oil refined for quality.

Once a canola line has been stabilized, fully conventional methods ofplant biotechnology, breeding and selection are used to further enhance,for example, the agronomic properties of the resultant line in order toimprove important factors such as yield; hardiness, etc. Such techniquesare also well known and include, e.g., somaclonal variation, seedmutagenesis, anther and microspore culture, protoplast fusion, etc. See,e.g., Brunklaus-Jung et al., Pl. Breed., 98:9-16, 1987; Hoffmann et al.,Theor. Appl. Genet., 61, 225-232 (1982), each herein incorporated byreference).

A deposit of seed designated IMC 01 has been made in the American TypeCulture Collection (ATCC) depository (Rockville, Md. 20852) and bearsaccession number ATCC 40579. The deposit was made on 2 Mar. 1989 underconditions complying with the requirements of the Budapest Treaty.

EXAMPLE 4

IMC 01 was compared to Alto, a “generic” variety of commercial canolaoil.

Oils were processed using standard commercial refining, bleaching anddeodorizing processes. TABLE X Processed Oil Analysis IMC 01 Alto Redcolor 0.5 0.7 Yellow color 4 5 para-anisidine value 0.97 2.32 Peroxidevalue, meg/g 0.6 0.7 TOTOX value - p-av + 2(pv) 2.07 3.72 TotalPolymers, % 0.02 0.01 Total Polar Material, % 0.77 0.36 Free fatty acid,% 0.022 0.013 Fatty acid composition, % C16:0 4.2 4.0 C18:0 2.2 2.0C18:1 63.5 62.5 C18:2 22.2 18.3 C18:3 4.9 7.7

Results: Chemical analysis indicates oils were processed within industrystandards for quality vegetable oils (see Table X). Based on AOCSRecommended Practice Cg 3-91; oils with a TOTOX value of less than 4.0indicate good stability. TOTOX value=para-anisidine value+2(peroxidevalue).

Colors determined by American Oil Chemists' Society (AOCS) method Cc13b-43, using American Oil Tintometer, Model AF715, The Tintometer LTD.,Salisbury, England.

para-Anisidine Value (p-av) determined by AOCS method Cd 18-90, measuressecondary oxidation by-producs in oils.

Peroxide Value determined by AOCS method Cd 8b-90, measures the primaryoxidation product in oils.

Total polymers determined by AOCS method Cd 22-91, gel-permeation HPLC.

Total Polar Materials determined by AOCS method Cd. 20-91, packed columnmethod adpated to HPLC.

Free fatty acid determined by AOCS method Ca 5a-40.

Fatty acid composition determined by AOCS method Ce le-91, capillary gasliquid chromatography.

Oxidative Stability by Oxidative Stability Instrument (OSI)

Oxidative stability measured by Automatic Oxygen Method (AOM) hoursdetermined by American Oil Chemists Society (AOCS) method Cd 12b-92, FatStability, Oil Stability Index (OSI), using an Oxidative StabilityInstrument, manufactured by Omnion/Archer Daniels Midland, Decatur, Ill.IMC 01 Alto AOM hours 26.8 17.5Results indicate IMC 01 has greater AOM hours and therefore greateroxidative stability than Alto.Oxidative Stability and Flavor Stability by Accelerated Aging

The Schaal oven method of accelerated aging is used in the oil industryto measure the oxidative and flavor stability of oil. The Schaal ovenmethod involves examining samples of oil at predetermined intervals heldat 60° C. in the dark. One day under Schaal oven conditions isequivalent to one month storage in the dark at ambient temperature.

Oxidative stability is measured by monitoring the increases in peroxides(peroxide value) and secondary oxidative by-products (para-anisidinevalue) in the oils held at 60° C. for twelve days.

Flavor stability is determined by a trained sensory panel using the sameoils tested for oxidative stability.

Sample Preparation:

400 g of oil placed in 500 mL amber glass bottles (80 wide, 140 mm high,with a 42 mm opening), uncapped, held in 60° C. (range 59 to 61° C.)convection oven (Blue M, manufactured by Blue M Electric) for 3, 6, 9and 12 days. One bottle of oil per day per type of oil.

Samples were frozen immediately after removing from the oven. Peroxidevalue, para-anisidine value and sensory evaluation were made withinseven days after samples were frozen.

Oxidative Stability by Accelerated Aging TABLE XI Changes in PeroxideValue and para-Anisidine Value PV p-AV Days IMC 01 Alto IMC 01 Alto 0 .60.7 0.97 2.32 3 0.9 5.33 0.99 2.52 6 5.48 11.8 1.36 5.02 9 10.3 15.72.45 6.5 12 14.1 18.8 3.42 7.49(see FIGS. 1 and 2)

Results: Based on increases in peroxide and p-av values over the 12 daystorage period, IMC 01 has greater oxidative stability than Alto (seeTable XI).

Flavor Stability by Accelerated Aging:

Sensory panel trained in evaluation of vegetable oils according toprotocol of AOCS method Cg 2-83, Flavor Panel Evaluation of VegetableOils. Using AOCS flavor standards panel members were trained to identifythe following off flavors; fishy, green, cardboard, plastic and painty.TABLE XII Overall Acceptance Scores and Total Off-Flavor IntensitiesOverall Acceptance¹ Total Off-Flavors² Day IMC 01 Alto IMC 01 Alto 07.53 6.01 0.59 2.32 3 7.04 3.2 0.87 8.29 6 5.44 3.10 4.17 9.46 9 3.811.78 6.22 10.15 12 4.00 2.00 7.16 12.89¹Overall acceptance scores are based on a 0-9 scale; 0 is extremelyunacceptable and 9 is very acceptable.²Total off-flavor intensities include fishy, green, cardboard, andpainty. Ratings are based on a 0-15 scale, were 0 is bland and 15 isvery intense.Results:

Overall acceptability scores were significantly different after 0, 3, 6,9, and 12 days (p=0.05) (see Table II).

Correlation between overall acceptance score and total off-flavorsr²−0.9485 (see FIG. 5).

Significantly lower overall acceptance scores and lower total off-flavorintensities indicates IMC 01 has significantly better flavor stabilitythan Alto.

EXAMPLE 5

IMC 02 was compared to Stellar, a commercially available low alphalinolenic variety of canola. TABLE XIII Processed Oil Analysis IMC 02Stellar Red color 0.2 0.3 Yellow color 3 3 para-Anisidine value 0.480.78 Peroxide value, meq/g 0.2 0.6 TOTOX value = p-av + 2(pv) 0.88 1.98Total Polymers, % 0.01 0 Total Polar Material, % 0.63 0.42 Free fattyacid, % 0.026 0.014 Fatty acid composition, % C16:0 3.8 4 C18:0 2.2 2.3C18:1 66.3 62.8 C18:2 22.3 24.4 C18:3 2.7 3.3Results: Chemical analysis indicates oils were processed within industrystandards for quality vegetable oils (see Table XIII). Based on AOCSRecommended Practice Cg 3-91; oils with a TOTOX value of less than 4.0indicate good stability. TOTOX value=para-anisidine value+2(peroxidevalue).

Oxidative Stability—OSI AOM Hours IMC 02 Stellar AOM hours 31.5 23.1

Results indicate IMC 02 has greater AOM hours and therefore greateroxidative stability than Stellar.

Oxidative Stability by Accelerated Aging TABLE XIV Changes in PeroxideValue and para-Anisidine Value PV p-AV Days IMC 02 Stellar IMC 02Stellar 0 0.2 0.6 0.48 0.78 3 0.7 1.39 0.76 0.84 6 1.5 3.77 0.26 0.99 94.4 7.37 0.82 2.07 12 9.6 13.3 1.18 2.56(See FIGS 6 and 7)

Results: Based on increases in peroxide and p-av values over the 12 daystorage period, IMC 02 has greater oxidative stability than Stellar (seeTable XIV). TABLE XV Overall Acceptance Scores and Total Off-FlavorIntensities Overall Acceptance¹ Total Off-Flavors² Day IMC 02 StellarIMC 02 Stellar 0 8.66 7.75 0.66 0.73 3 6.87 4.18 1.98 7.16 6 7.01 4.571.44 6.07 9 6.10 3.67 2.79 8.41 12 4.16 3.67 5.73 7.83¹Overall acceptance scores are based on a 0-9 scale; 0 is extremelyunacceptable and 9 is very acceptable.²Total off-flavors include fishy, green, cardboard, plastic and painty.Ratings are based on a 0-15 scale, where 0 is bland and 15 is veryintense.(See FIGS. 8 and 9)

Results: Overall acceptability scores were significantly different after0, 6 and 9 days (p=0.05) (see Table XV).

Correlation between overall acceptance score and total off-flavorintensities is r²−0.9428 (see FIG. 10).

Significantly lower overall acceptance scores and lower off-flavorintensities indicates IMC 02 has signficantly better flavor stabilitythan Stellar.

1. A canola plant line designated IMC 02 and having American TypeCulture Collection (ATCC) Accession No. PTA-6221.
 2. Progeny of thecanola plant line of claim 2, said progeny producing seeds having anα-linolenic acid content of from about 1.6 to about 7% and an aliphaticand indolyl glucosinolate content of about 13 μmol or less per gram ofdeFatted, air-dried meal.
 3. The progeny of claim 2, wherein saidα-linolenic acid content is from about 1.9% to about 4.1%.
 4. Seeds ofthe progeny of claim 2, said seeds having said α-linolenic acid contentand said aliphatic and indolyl glucosinolate content.
 5. Plants of theprogeny of claim 2, seeds of said progeny having said α-linolenic acidcontent and said aliphatic and indolyl glucosinolate content.
 6. Canolameal extracted from the seeds of claim 2, said canola meal having analiphatic and indolyl glucosinolate content of about 13 μmol or less pergram of defatted, air-dried meal.
 7. The progeny of claim 2, whereinsaid α-linolenic acid content is from about 1.6 to about 1.8% seeds havean ______
 8. Seed of a Brassica napus canola plant line, said seedhaving an α-linolenic acid content of from about 1.6 to about 7% and analiphatic and indolyl glucosinolate content of about 13 μmol or less pergram of defatted, air-dried meal, wherein said plant is a descendant ofa cross of a plant line designated IMC 02 and having ATCC Accession No.PTA-6221 with a second non-IMC 02 Brassica napus line.
 9. Progeny of theseed of claim 8, said progeny producing seeds having an α-linolenic acidcontent of from about 1.6% to about 7% and an aliphatic and indolylglucosinolate content of about 13 μmol per gram of meal.
 10. The seed ofclaim 8, wherein said α-linolenic acid content is from about 1.9% toabout 4.1%.
 11. Canola meal extracted from the seed of claim 8, saidcanola meal having an aliphatic and indolyl glucosinolate content ofabout 13 μmol or less per gram of meal.
 12. The seed of claim 8, whereinsaid α-linolenic acid content is from about 1.6% to about 2.8%.
 13. Theseed of claim 8, having an aliphatic glucosinolate content of about 3.7to about 6.3 μmol per gram of meal.
 14. The seed of claim 8, whereinsaid seed has an average aliphatic glucosinolate content of about 4.9μmol per gram of meal.